Shigella dysenteriae type 1 and strains of Escherichia coli which produce, respectively, the protein exotoxins Shiga toxin and Shiga-like toxins are responsible for a variety of diseases in humans and animals. In humans, toxin-producing bacteria cause colonic disease which may progress to hemorrhagic colitis (15, 17) and serious extraintestinal sequelae, including thrombotic thrombocytopenic purpura (18) and the hemolytic uremic syndrome (11, 15). Weaned pigs infected with E. coli producing Shiga-like toxin II variant (SLT-IIv) develop edema disease (ED), a disorder characterized by profound generalized vascular damage, edema in subcutaneous and gastrointestinal tissues, neurological abnormalities, and death (2, 16).
Strains of E. coli that cause ED are included within a limited number of serogroups, identified as 0138, 0139, and 0141 (10). These strains can produce several proteins which may act as virulence factors in vivo, including heat-labile enterotoxin, heat-stable enterotoxin, hemolysin, and SLT-IIv. Recently, MacLeod et al. reported that intravenous injection of pigs with purified SLT-IIv at doses as low as 1.5 ng/kg of body weight resulted in the development of lesions which are characteristic of ED (14). Therefore, despite the presence of other putative virulence factors in ED isolates, intoxication with SLT-IIv appears to account for the majority of signs seen in ED.
All members of the Shiga-like toxin family are holotoxins, composed of a single A subunit (32.2 kDa) in noncovalent association with a pentamer of B subunits (7.7 kDa) (20, 21). Enzymatic activity resides in the A subunit, and binding activity is conferred by the B subunit. The A subunit is a specific N-glycosidase which cleaves a single adenine from the 28S rRNA component of eukaryotic ribosomes (3). This action results in an inhibition of protein synthesis.
Although antigenically similar to SLT-II (16), SLT-IIv is classified as a variant because it has reduced toxicity for HeLa cells, whereas SLT-II is equally toxic to both Vero and HeLa cells (16). This differential cytotoxicity may be explained, in part, by a difference in eukaryotic receptor binding. Although both toxins bind glycolipid of the globo-series, SLT-IIv appears to preferentially bind globotetraosylceramide (Gb.sub.4), while SLT-II and other members of the Shiga-like toxin family primarily bind globotriaosylceramide (Gb.sub.3) on the target cell surface (22). The nucleotide sequence and the deduced amino acid sequence for the A and B subunits of SLT-IIv are known (26).
Currently, there are no effective vaccines against ED. Using oligonucleotide-specific site-directed mutagenesis, we have identified amino acid changes in the A subunit that drastically reduce the enzymatic activity of SLT-IIv. Such a mutagenized SLT-IIv represents a suitable candidate for a vaccine against ED.